Our long-term objective has been the study of the molecular/cellular mechanisms for the initiation and promotion phases of carcinogenesis using a variety of in vitro approaches. In this proposal, we plan to focus entirely on the promotion/progression phases of carcinogenesis. The aim is to test the specific hypothesis that gap junctional intercellular communication (GJIC) might play a role in tumor promotion and possibly the progression phase of carcinogenesis. The rationale supporting this aim is based on the observations that: (a) most, if not all, malignant cells have altered selective or universal gap junctional communication; (b) most known chemical tumor promoters have been shown to down-regulate gap junction function, in a reversible fashion; (c) several oncogenes (e.g., src, ras, raf, neu, mos, but not myc) are associated with stable down-regulation of GJIC; (d) several anti-tumor promoters or anti-tumor agents (e.g., retinoids and lovastatin) are associated with the MR-regulation of GJIC, and (e) several tumor suppressor genes have been linked to the up-regulation of GJIC. Therefore, we plan to test several hypotheses for the biochemical mechanisms by which two different classes of chemical tumor promoters (phorbol ester; DDT), a few oncogenes (ras and neu), tumor suppressor genes (Stanbridge's human suppressor gene) and anti-tumor promoters or anti-tumor agents (e.g., lovastatin; retinoids) might modulate gap junction function. In addition, in order to directly test the hypothesis that GJIC plays a role in carcinogenesis, an attempt will be made to restore GJIC in GJIC-deficient and tumorigenic rat liver epithelial cell mutants by transfection with several cloned gap junction genes in various expression vectors. We will also transfect GJIC proficient, non-tumorigenic rat liver cells with a gap junction anti-sense gene to test the hypothesis that the specific loss of functional the gap junction message will result in a non-communicating cell which will be tumorigenic when placed back in the rat liver. To accomplish these goals, various biological, biochemical, and molecular techniques available in our laboratory (e.g., fluorescence redistribution after photobleaching, scrape-loading/dye transfer assays, molecular/antibody probing, using cDNAs and antibodies to various gap junction genes and proteins, DNA cloning, amplification, cloning and transfer, etc.) will be employed.